Determinants of DNA binding and bending by the Saccharomyces cerevisiae high mobility group protein NHP6A that are important for its biological activities. Role of the unique N terminus and putative intercalating methionine.
The non-histone proteins 6A/B (NHP6A/B) of Saccharomyces cerevisiae are high mobility group proteins that bind and severely bend DNA of mixed sequence. They exhibit high affinity for linear DNA and even higher affinity for microcircular DNA. The 16-amino acid basic segment located N-terminal to the high mobility group domain is required for stable complex formation on both linear and microcircular DNA. Although mutants lacking the N terminus are able to promote microcircle formation and Hin invertasome assembly at high protein concentrations, they are unable to form stable complexes with DNA, co-activate transcription, and complement the growth defect of Deltanhp6a/b mutants. A basic patch between amino acids 13 and 16 is critical for these activities, and a second basic patch between residues 8 and 10 is required for the formation of monomeric complexes with linear DNA. Mutational analysis suggests that proline 18 may direct the path of the N-terminal arm to facilitate DNA binding, whereas the conserved proline at position 21, tyrosine 28, and phenylalanine 31 function to maintain the tertiary structure of the high mobility group domain. Methionine 29, which may intercalate into DNA, is essential for NHP6A-induced microcircle formation of 75-bp but not 98-bp fragments in vitro, and for full growth complementation of Deltanhp6a/b mutants in vivo.
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Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095-1737, USA.
Wong
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Johnson
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D000595:Amino Acid Sequence; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024001:HMGN Proteins; D007364:Intercalating Agents / Q000378:metabolism; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D024001:HMGN Proteins; D007364:Intercalating Agents; C086471:NHP6A protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D008715:Methionine